Thymoma‐associated autoimmune encephalitis with myasthenia gravis: Case series and literature review

Abstract Objectives This comprehensive review aimed to compile cases of patients with thymoma diagnosed with both autoimmune encephalitis (AE) and myasthenia gravis (MG), and describe their clinical characteristics. Methods Clinical records of 3 AE patients in the first affiliated hospital of Sun Yat‐sen University were reviewed. All of them were diagnosed with AE between 1 November 2021 and 1 March 2022, and clinical evidence about thymoma and MG was found. All published case reports were searched for comprehensive literature from January 1990 to June 2022. Results A total of 18 cases diagnosed with thymoma‐associated autoimmune encephalitis (TAAE) and thymoma‐associated myasthenia gravis (TAMG) were included in this complication, wherein 3 cases were in the first affiliated hospital of Sun Yat‐sen University and the other 15 were published case reports. 5/18 patients had alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor antibody (AMPAR‐Ab) in their serum and cerebrospinal fluid (CSF). All of them had positive anti‐acetylcholine receptor antibody (AChR‐Ab). And 12/18 patients showed a positive response to thymectomy and immunotherapy. Besides, thymoma recurrences were detected because of AE onset. And the shortest interval between operation and AE onset was 2 years in patients with thymoma recurrence. Conclusions There was no significant difference in the clinical manifestations between these patients and others with only TAMG or TAAE. TAAE was commonly associated with AMPAR2‐Ab. Significantly, AE more commonly heralded thymoma recurrences than MG onset. And the intervals of thymectomy and MG or AE onset had different meanings for thymoma recurrence and prognoses of patients.


| INTRODUC TI ON
Thymoma can occur with autoimmune disorders since it originates from epithelial cells of the thymus, the important immune organ for the maturation and differentiation of T-lymphocytes. 1 And the most common thymoma-associated autoimmune disease is myasthenia gravis (MG), which occurs in 30% of patients with thymoma. 2 MG is characterized by muscle weakness and fatigability due to the antibodies against autoantigens at the neuromuscular junction (NMJ).And while the main autoantibodies of thymoma-associated MG (TAMG) against skeletal muscle are anti-acetylcholine receptor antibodies (AChR-Abs), the clinical manifestations have great heterogeneity. 2 Thymoma-associated autoimmune diseases also include autoimmune encephalitis (AE).But AE is an uncommon disease compared with MG according to a multicenter and retrospective study, in which only 1 patient had thymoma-associated AE (TAAE) in 85 patients with thymoma. 3AE is a neurological disorder which presents with cerebral inflammation involving gray matter with or without white matter, caused by neuronal cell antibodies or intracellular antibodies. 4The most common antibody of thymoma-associated AE was gamma-aminobutyric acid receptor A antibody (GABA A R-Ab), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antibody (AMPAR-Ab) secondly. 5evious series 5,6 of thymoma-associated autoimmune diseases usually focused on the analysis of clinical features and autoantibody repertoire in patients with only thymomatous MG or thymomatous AE.Moreover, there were only some case reports for the clinical manifestations of patients with overlapping of TAMG and TAAE, which lacked systematic description.The objective of this article was to describe the sequence of MG and AE, the clinical presentations, the examination results and the management, as well as the prognosis of 3 patients with TAAE and TAMG from our center and conduct a comprehensive literature review with another 15 cases identified from previous case reports and case series.

| Case series
The clinical details of three patients with TAAE and TAMG from our institution were reviewed, and case 2 was diagnosed with AE elsewhere.In all cases, the clinical presentation, including memory impairments, confusion and abnormal behaviors and so on, brain MRI, CT of the chest, CSF analysis and cytology, immunosuppressive therapy, surgery, pathological classification of thymomas, and prognosis were recorded.

| Review of the literature
Search terms "thymoma," "autoimmune encephalitis," and "myasthenia gravis" identified 15 case reports through Embase, MEDLINE, and Scopus databases from January 1990 to June 2022.The references of articles which were identified by these search terms also were filtered for more relevant articles.The clinical presentation, brain MRI, autoimmune antibodies test, immunosuppressive therapy, types of surgery, pathological classification of thymomas, and prognosis in these case reports were recorded in Table 1 and   The repetitive nerve stimulation (RNS) of electromyography and neostigmine test were unable to be completed due to the progressive symptoms of this patient.
Intravenous immunoglobulin (IVIg) 0.4 g/(kg*day) for 5 days and methylprednisolone 40 mg/day were given.But the symptoms did not improve after immunotherapy.On the 4th day after admission, he was in a light coma and a continued hypoxemia because of the disease progression and the pulmonary infection.An urgent trachea intubation was performed and the respirator was used to support his respirations.The thoracic surgeons and the radiation oncology physicians suggested that there was no indication for further surgery, chemotherapy, or radiotherapy.The patient was in a consistent coma and presented unstable blood circulation which needed norepinephrine to stabilize blood pressure.The family finally gave up and the patient died soon after discharge from day 14 after admission.

| Case 2
A 39-year-old woman underwent surgery following the diagnosis of thymoma and the histopathology showed type B1 thymoma.She developed generalized MG with fluctuated weakness, which included  On the 1st day after admission, the patient was agitated and had violent and self-injury behaviors.In a state of great agitation, he suddenly went into a coma, and then his carotid pulse disappeared, his nail bed and lips were cyanosed, and the oxygen saturation was too low to be measured.Chest compressions, ventilation with the balloon, and an intramuscular injection of epinephrine His family finally gave up and the patient died soon after discharge from day 22 after admission.

| Literature review
Over the past 32 years, we identified 18 patients who had been diagnosed with thymoma, MG, and AE, including 15 cases that have been reported, and of them, 11 (61.11%) were female.MG and AE were diagnosed concomitantly in 9/18 patients (50%).The median age at thymoma diagnosis was 44.5 years, from age 19 to 69.The earliest case was reported by Kodama et al. 7 in 1991, in which a 34-year-old woman was found to have a thymoma after presenting with AE and MG symptoms at the same time.Due to the limitation of detection technique, AE-associated antibody was not detected in this patient.
All the features of patients and their diseases are summarized in Table 1 and Table S1.
Our results showed that 10/18 patients (55.56%) had MG onset earlier than AE, and in these patients, the interval between MG and AE was from 1 month to 7 years with a mean interval of 2.335 years and a median interval of 1.5 year.6/18 patients (33.33%) were diagnosed with MG and AE concomitantly, while 2/18 patients (11.11%) presented AE symptoms at first, including one patient with an interval of 18 years and the other patient with an interval of 8 months.
In 18 patients, the most common skeletal muscle group affected by MG was ocular muscle (15/18, 83.33%), followed by bulbar muscle (9/18, 50%).The other muscle groups involved respiratory TA B L E 1 Clinical characteristics of 18 patients with TAAE and TAMG in English case reports.

| DISCUSS ION
Cases in this study presented the clinical manifestations in patients diagnosed with thymoma with MG and AE and provided more information on these patients for clinical decisions.We found that the features of MG and AE in these patients were similar to TAMG and TAAE.However, the intervals between thymectomy and MG or AE onset had different meanings for thymoma recurrence and of patients.
Our study found that patients diagnosed with thymoma with both MG and AE were most frequently associated with ocular muscle weakness, followed by bulbar muscles and respiratory muscles.Nearly all patients had detectable AChR antibodies, but no MuSK (MG patients have antibodies against the muscle-specific kinase, MuSK) or LRP4 (MG patients have antibodies against the agrin receptor low-density lipoprotein receptor-related protein 4, LRP4), similar to studies about TAMG. 6Initial studies described that in 10%-20% of MG patients, thymoma occurred and the age range of such patients was 40-60 years. 2,21varez-Velasco et al. have found that more frequent generalized symptoms and myasthenic crises occurred in patients with thymomatous MG, for which these patients were worse in their prognosis even after thymectomy. 6Besides, these patients with positive AChR IgG might also have RYR IgG or Titin IgG in serum tests, such as patient 1.
On the one hand, anti-AChR Abs cause disruption of neuromuscular junction (NMJ) components' signaling 21 by recognizing and attacking extracellular epitopes of the post-synaptic membrane, which leads to AChR degradation.[24][25]

Lymphocytic pleocytosis and protein level elevation
The patients in our review presented memory impairment, confusion, and seizures most commonly in AE, which were consistent with the clinical picture of limbic encephalitis (LE). 4LE, a specific clinical syndrome of AE with a clinical picture consisting of memory deficit, confusion, mood changes, and seizures, 4,26 was first described in 1960 27 and was replaced by term AE in referring to the disease entity since the autoimmune etiology has been recognized. 28Although the MRI of brain seemingly failed to show consistent and specific abnormalities in AE patients, temporal lobes were highly involved, which also met the MRI criteria of LE in the previous study. 4Furthermore, the symptoms and the abnormalities of brain MRI are closely linked to the AE-associated antibodies.
Antibodies which mediate AE are categorized into two types: (a) neuronal cell surface antibodies more frequently found, and (b) intracellular antibodies based on the antigens that they targeted. 29In our studies, patients more commonly had positive AMPAR2-Ab or VGKC-Ab.It is important to note that antibodies previously considered to directly bind to the voltage-gated potassium channel (VGKC) were discovered to bind to other proteins in the VGKC complexes including LGI1, CASPR2, and Contactin-2. 30Based on observations over recent years, there were 3 autoantibodies of AE associated with thymoma involving anti-AMPAR antibody, anti-GABA A R antibody, and anti-LGI1 antibody, which mainly targeted the limbic system in antibodies-mediated AE. 31 Anti-AMPAR AE is usually described with typical limbic encephalitis and a high rate of relapse with or without tumor recurrences. 32,33AMPARs are glutamate-gated ion channels composed of GluR-1, -2, -3, and -4 subunits.And alternative splicing and RNA editing of subunits result in different isoforms of AMPARs. 34Anti-AMPAR IgG antibodies frequently targeting the GluR1/2 subunits, internalize and degrade the receptors and lead to a global decrease in AMPA receptors of the synapse. 32Since GluR1/2 and GluR2/3 have the highest level of expression in the hippocampus and are associated with spatial learning and memory, 34 patients with anti-AMPAR encephalitis had memory loss and presented clinical manifestations of LE in the review.Although anti-GABA B R AE is more likely to occur with small-cell lung cancer (SCLC), case 3 had positive GABA B R-Ab. 35 In the case presented by Alexopoulos et al., 36 the strong expression of GABA B R in the thymic epithelial cells had been proven by immunocytochemistry in the patient with thymomaassociated anti-GABA B R encephalitis.GABA B Rs, comprised of GB1 and GB2 subunits, are G protein-coupled receptors for GABA (one kind of inhibitory neurotransmitter). 37Anti-GABA B R antibodies bind the GB1 subunit and block the function of GABA B R. 38 Although GB1 subunit are detected in the central nervous system (CNS) widely, the regions with the greatest level of GABA B R are hippocampus, 39 which are similar to AMPARs.Being different from anti-AMPAR encephalitis, anti-GABA B R encephalitis is more often associated with seizure and status epilepticus 40 because GABA B Rs mediate the inhibition of neuron unit discharges. 38,39In addition, low titers of anti-NMDAR antibodies can be additional antibodies of other CNS disorders possibly based on the opening of the blood-brain barrier. 31e reversibility of internalization of neuron membrane receptors or function-blocking of epitopes offers a possible explanation for the effectiveness of treatments that remove the pathological antibodies, such as intravenous immunoglobulin, plasma exchange, and immunoadsorption.The prognosis of thymoma-associated AE is dictated by thymoma recurrences.
Patients with TAAE were mostly normal in CSF blood cell and protein examinations.The presence of thymoma-associated autoimmune complications is associated with the types of thymic pathology.For instance, autoimmune diseases are more common in patients with WHO B and AB thymoma subtypes. 42Histologic subtypes AB, B1, and B2 were highly associated with MG since thymoma actively exports more autoreactive T cells in these subtypes. 2,43The expression of self-antigens by thymic epithelial cells 1,36 and the disorder of self-tolerance T-cell selection through the thymus 44 are considered as the link between tumors of the thymus and autoimmune complication.Then, the autoreactive T cells affect B cells and activate humoral immunity which further induces autoimmune diseases. 2As autoantibodies are produced in peripheral blood and can be independent of thymoma due to the existence of long-lived plasma cells 21 and long-memory T lymphocytes, 42,45 thymoma resection does not mean remission of autoimmune diseases.MG is difficult to be completely cured because as Buckley et al found, mature and autoreactive T cells, which induce autoantibody production in MG, remained in peripheral blood for over 20 years following thymectomy. 45Early-onset post-thymoma MG (<6 months after thymectomy) is supposed to be mediated by extrathymic antibody production and late-onset post-thymoma MG (≥6 months) may correlate with thymoma recurrence. 46There was no difference in the effectiveness of thymectomy on prognosis being found between pre-thymoma MG and post-thymoma MG, 46 but the long-term clinical benefit has been proven through reductions in Quantitative Myasthenia Gravis scores and increase in patients who reached the minimal manifestation status. 47Patients with TAMG usually get clinical remission depending on anticholinesterase and immunosuppressants.
Compared with MG, AE is more possibly to be completely cured after thymectomy and immunotherapy without thymoma local invasion or metastasis in our studies.However, paraneoplastic AE with onconeuronal antibodies rarely responds to immunotherapy 48 since the pathogenic mechanism mainly consists of cellular immunity mediated by cytotoxic T cells which needs to be distinguished from paraneoplastic AE with neuronal cell surface antibodies. 49In a retrospective cohort study conducted by Guasp and his colleagues, antibodies against intracellular antigens only occur in 30% of patients with thymoma-associated AE. 5 And concurrent with onconeuronal antibodies means poor outcomes among patients with anti-AMPAR encephalitis. 5Previous studies also emphasized the importance of surgical removal of thymoma along with immunotherapy in TAAE. 33,50It also reminds us of different immunopathogenesis between MG and AE.Although the mechanism was still unclear, we supposed that AE was more likely to be mediated by thymoma and thymoma recurrence rather than extrathymic antibody production.
In our studies, AE arising after several years following thymectomy indicated a worse prognosis since it has more possibility to herald a tumor recurrence than MG.The long interval between thymectomy and AE onset, and the recurrence of tumor can be used to explain the poor outcome of patient 1 compared with patient 2. The research of Guasp et al also showed that 30% of TAAE had a previous diagnosis of MG and 51% of TAAE was complicated with thymoma invasion or metastasis. 5And MG onset often led to thymoma detection and correlated with early Masaoka stage (I and II). 3 On the other hand, neurotropic virus infections may trigger AE through polyclonal immunoreaction, 51,52 which means antibodies are produced in polyspecific immune response against non-causative antigens of CNS.
The association between herpes virus family and neurological autoimmune disease has been described in several research, such as HSV and AE, 53 EBV and multiple sclerosis (MS), 52 EBV and AE. 51Since 3 gene sequences of EBV were detected in CSF of patient 1, the elapsed infection of EBV might also contribute to the pathogenesis of AE.However, the role of elapsed CNS infection in thymomaassociated AE is still unknown.
The myogenic damage showed by the electromyography suggested that the patient in case 2 might have simultaneous onset of to thymoma according to the case series reported by Huang et al. 54 And the abnormal T-cell activation is part of the potential pathogenesis of both MG and IM. 55Other thymoma-associated autoimmune diseases include thyroid dysfunction, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), alopecia areata, pure red cell aplasia (PRCA), polymyositis, and Good's syndrome, and so on. 3,56Thyroid dysfunction was the most frequent autoimmune disorder except analyzed in Tables S1, S2 and Figure S1 in the supplementary material.
year-old male accepted thymoma resection after thymoma detection in December 2018, and the histopathological classification was type B2 (World Health Organization thymoma classification).Before the thymectomy, he had no symptoms of MG or AE.But the patient had short-term memory loss and confusion and became unresponsive after 3 years following thymectomy in December 2018.The disease progressed so rapidly that the patient could not walk due to the fatigable weakness of the bilateral leg, and had dysphagia and incontinence.The chest computed tomography (CT) revealed that the thymoma recurred, metastasized, and even invaded the pericardium.His brain MRI with Gd-enhancement was normal (Figure 1A,1C).MG and AE were diagnosed concomitantly since serum antibody tests showed positive ACh-R IgG (8.014 nmol/L), RyR (ryanodine receptor) IgG, Titin IgG, and AMPAR2-Ab (1:32), while CSF AE antibody tests showed positive AMPAR2-Ab (1:20).The pathogen Next Generation Sequencing (NGS) of Cerebrospinal Fluid (CSF) showed 3 human herpes virus type 4 (Epstein-Barr virus, EBV) gene sequences.The other cerebrospinal fluid (CSF) examination was normal except for a lymphocytic pleocytosis of 41 cells/mm 3 (normal: <5 cells/ mm 3 ).Besides, raised thyroid peroxidase antibodies (TPOAb) and thyroglobulin antibodies (TgAb), as well as decreased free thyroxine (fT4) and free triiodothyronine (fT3) were present with thyroid stimulating hormone (TSH) in the thyroid function assessment.

| 3 of 12 SU
et al. left ptosis, dysphagia, dysarthria, breathing disorders, and bilateral brachial weakness with swelling and pain over 10 days after the operation.Her muscle weakness was alleviated in the morning or after rest.And the patient had a positive ocular fatigue test and a good response to cholinesterase inhibitor therapy but a negative neostigmine test.Furthermore, her serum antibody test showed positive ACh-R IgG.The electromyography showed myogenic damage, normal RNS, and an elevated serum muscle enzyme level.But she still had fatigable weakness after her serum muscle enzyme level declined to a normal level.the CT of the chest was normal without any thymoma recurrence or residual tumor.She was treated with methylprednisolone 40 mg/day and intravenous immunoglobulin (IVIg) 0.4 g/(kg*day) for 5 days.Then her symptoms of MG improved.She received radiotherapy after remission and continued taking methylprednisolone (25 mg once a day) and pyridostigmine bromide (60 mg 3 times a day) for MG.The onset of AE occurred 3.6 years after the operation since the patient stopped taking methylprednisolone for 2 months.She experienced short-term memory loss and repeatedly checked the time and place of her appointment with her friends.Besides, confusion and alopecia areata, which included localized hair loss of the head and pubis, occurred.MRI of the brain with Gd-enhancement was normal (Figure 1B,1D).CT of the chest and PET imaging were performed and no signs of malignancy recurrence was suspected.The electroencephalogram (EEG) was normal.The serum antibodies testing showed positive AMPAR-Ab (1:32).CSF revealed positive AMPAR-Ab (1:32) and positive anti-N-methyl-D-aspartate receptor antibody (NMDAR-Ab) (1:3.2).Normal in the CSF were the CSF pressure, white blood cell (WBC), protein, glucose, chloride, and bacteriological culture.She accepted IVIg 0.4 g/(kg*day) for 5 days and corticosteroid impulse therapy (methylprednisolone 500 mg/ day for 3 days and follow-up oral prednisone 50 mg/day).She was discharged since her memory was reversed.Nine months later, the patient went back for further assessment.The serum antibody test revealed negative Ach-R IgG and NMDAR-Ab, but positive Titin IgG and AMPAR-Ab (1:32).And she had lower AMPAR-Ab (1:1) in CSF.Neuropsychological testing showed a mini-mental state examination (MMSE) of 30/30.The testing related to MG showed a quantitative myasthenia gravis score (QMGS) of 2, a myasthenia gravis-activities of daily living profile (MG-ADL) of 100, and a modified Rankin Scale (mRS) of 1/5.Now, her daily activity is compatible with before.

3. 1 . 3 | Case 3 A 49 -
year-old man presented to our neurological clinic in January 2022 with fluctuated bilateral ptosis, visual hallucination, and delusion of persecution.His ptosis was alleviated in the morning.CT of the chest showed an anterior superior mediastinal mass, considering the thymic tumor and left pleural metastasis.The serum antibodies testing showed positive GABA B R-Ab (1:100).CSF revealed positive GABA B R-Ab (1:100).His MRI of brain with Gd-enhancement was normal.The neostigmine test was negative.MG's proof of diagnosis is a positive ACh-R IgG serum test (14.57nmol/L).The CSF analysis showed protein level elevation of 70 mg/dL (normal: <50 mg/ dL).Normal investigations included CSF pressure, glucose, chloride, cell count, and bacteriological culture.No tumor cell was detected in CSF.The repetitive nerve stimulation (RNS) of electromyography was unable to be completed due to the progressive symptoms of this patient.

( 1
mg) were given for emergency treatment and the patient's spontaneous respiration was restored in 3 minutes.After tracheal intubation, he accepted further treatment in the intensive care unit (ICU) with mechanical ventilation.The symptoms persisted despite increasing immunosuppressive medication including methylprednisolone impulsion 1 g/day for 3 days and subsequently 40 mg/day, tacrolimus 2.184 mg/day, intravenous immunoglobulin (IVIg) 0.4 g/ (kg*day), and plasma exchanges for 5 days.The thymoma resection was performed on the 14th day after admission and the histopathology was sclerosing thymoma.Postoperative CSF GABA B R-Ab titer decreased (1:1+), but serum GABA B R-Ab remained (1:100) and psychiatric symptoms and muscle weakness were not improved.

F I G U R E 1 | 9 of 12 SU
(A) T2-FLAIR images of the brain MRI of patient 1 after AE onset showed no abnormal hyperintensity.(B) T2-FLAIR images of the brain MRI of patient 2 after AE onset showed no abnormal hyperintensity.(C) Lack of enhancement of the brain MRI of patient 1 after AE onset.(D) Lack of enhancement of the brain MRI of patient 2 after AE onset.(Brain MRI images of patient 3 after AE onset cannot be accessed but its word report showed no abnormal hyperintense.)More images can be found in Figure S2.et al.
MG and inflammatory myositis (IM).The fluctuated muscle weakness with the normal level of serum muscle enzymes, the well therapeutic responses to cholinesterase inhibitor, and the positive serum AChR Abs still supported the diagnosis of MG in case 2. The diagnosis of inflammatory myopathy (IM) was unclear due to the lack of medical records about positive antibodies related to IM.It seems that MG or IM is typically present solely.But the co-existence of the diseases is a concomitant inflammatory phenomenon related 41ith 9/18 patients in our review, mild lymphocytic pleocytosis (usually <100/mm 3 ) or protein level elevation (usually <170 mg/dL) could also occur in over/approximately 50% of patients with paraneoplastic encephalitis41and be explained by inflammatory changes.CSF analysis assists clinicians in making the diagnosis of immune-mediated neurological diseases and the differential diagnosis including central nervous system infections and leptomeningeal metastases.But there was no evidence of a connection between these results of CSF and the prognosis of patients.